The ADP-ribosylation factors (ARFs) are a family of GTP binding proteins that regulate membrane traffic and organelle structure in the cell. We have been studying the cellular function of ARF6. Using transient transfection of HeLa cells expressing wild type and mutants of ARF6, we have shown that ARF6 regulates, through its GTP cycle, the movement of plasma membrane (PM) into and back out of a novel, endosomal compartment. ARF6-GTP at the plasma membrane (PM) stimulates the formation of actin-rich protrusions, and upon GTP hydrolysis, ARF6-GDP and membrane is internalized, to form the recycling endosome; recycling of membrane back to the PM requires activation of ARF6. This ARF6-regulated, novel recycling pathway exists in the absence of ARF6 overexpression, and endogenous PM proteins such as MHC class I, are found to cycle through it. This membrane traffic pathway and its effect on actin polymerization may be utilized to alter cell shape. In support of this, we have identified a requirement for ARF6 in two instances. 1) Activated Rac1, another GTP binding protein, induces the formation of membrane ruffles, distinct from ARF6-induced protrusions, over the surface of cells. Rac-stimulated ruffling is dependent upon activation of ARF6 and the functioning of its regulated membrane recycling pathway. In contrast, ARF6-stimulated protrusions form independently of Rac activity. 2) Endogenous ARF6 is localized in a number of cell types to internal, endosomal structures, and at the edges of cells, at sites of cortical actin filaments and membrane ruffling. During spreading of rounded cells onto culture dishes, cells form protrusions containing ARF6 and actin. Formation of these protrusions and cell spreading is inhibited when the dominant negative, T27N mutant of ARF6 is expressed in these cells, thus implicating ARF6 and its regulation of membrane recycling for cell spreading.